Fluorescence based flow imaging and measurements

ABSTRACT

Fluorescence based tracking of a light-emitting marker in a bodily fluid stream is conducted by: providing a light-emitting marker into a fluid stream; establishing field of view monitoring by placement of a sensor, such as a high speed camera, at a region of interest; recording image data of light emitted by the marker at the region of interest; determining time characteristics of the light output of the marker traversing the field of view; and calculating flow characteristics based on the time characteristics. Furthermore generating a velocity vector map may be conducted using a cross correlation technique, leading and falling edge considerations, subtraction, and/or thresholding.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation U.S. Non-Provisional patentapplication Ser. No. 15/863,338, entitled “FLUORESCENCE BASED FLOWIMAGING AND MEASUREMENTS,” filed on Jan. 5, 2018, which is acontinuation of International Patent Application Serial No.PCT/US2016/041045, entitled “FLUORESCENCE BASED FLOW IMAGING ANDMEASUREMENTS,” filed on Jul. 6, 2016, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 62/189,126, entitled“FLUORESCENCE BASED FLOW IMAGING AND MEASUREMENTS,” filed on Jul. 6,2015. Each above-reference application is incorporated herein in itsentirety by this reference.

TECHNICAL FIELD

The present disclosure relates to fluorescence based imaging andmeasurements. More particularly, the present disclosure relates todetermining flow characteristics such as velocity in bodily vessels suchas blood vessels.

BACKGROUND

Fluorescent markers have been used for basic imaging of bodilystructures, but improvements are needed in determining flowcharacteristics in such bodily fluids as blood.

SUMMARY

This summary is provided to introduce in a simplified form concepts thatare further described in the following detailed descriptions. Thissummary is not intended to identify key features or essential featuresof the claimed subject matter, nor is it to be construed as limiting thescope of the claimed subject matter.

According to at least one embodiment, a method of fluorescence basedtracking of a light-emitting marker in a bodily fluid stream includes:providing a light-emitting marker into a bodily fluid stream;monitoring, with a sensor, a region of interest traversed by the bodilyfluid stream; recording data generated by the sensor; determining timecharacteristics of the recorded data; and calculating flowcharacteristics based on the time characteristics.

In at least one example, the sensor includes a camera, and the recordeddata comprises motion video data.

In at least one example, the method further includes: dividing themotion video data into kernels; identifying which of the kernels receivesome portion of the light-emitting marker using an intensity threshold;computing, for each identified kernel, an intensity signal data setincluding information of mean light intensity versus time; performingsmoothing on each intensity signal data set; calculating a lag timebetween the intensity signal data sets of neighboring identified kernelsusing cross-correlation; using a spatial resolution and the lag time,calculating velocity vectors; summing the velocity vectors ofneighboring kernels to create a resultant velocity vector; andgenerating a velocity map from the resultant velocity vectors for allkernels.

In at least one example, performing smoothing on each intensity signaldata set includes time window averaging.

In at least one example, performing smoothing on each intensity signaldata set includes using a filter.

In at least one example, wherein performing smoothing on each intensitysignal data set includes using a Gaussian filter.

In at least one example, the method further includes: dividing themotion video data into kernels; identifying which of the kernels receivesome portion of the light-emitting marker using an intensity threshold;computing, for each identified kernel, an intensity signal data setincluding information of mean light intensity versus time; performingsmoothing on each intensity signal data set; for each particularidentified kernel, finding segments in which a slope of the intensitysignal data set rises for a minimum consecutive number of frames orfalls for a minimum consecutive number of frames, which segments occurwhen a leading edge or falling edge of a portion of the light-emittingmarker passes through the identified kernel; searching the intensitysignal data sets of neighboring identified kernels for a rising orfalling segment of similar length; calculating a lag time betweensegments in the particular identified kernel and segments in theneighboring identified kernels; using a spatial resolution and the lagtime, calculating velocity vectors; summing the velocity vectors ofneighboring kernels to create a resultant velocity vector; andgenerating a velocity map from the resultant velocity vectors for allkernels.

In at least one example, performing smoothing on each intensity signaldata set includes time window averaging. In at least one example,performing smoothing on each intensity signal data set includes using afilter. In at least one example, performing smoothing on each intensitysignal data set includes using a Gaussian filter.

In at least one example, the method further includes: calculating adifference frame by subtracting a frame of the motion video data from aconsecutive frame of the motion video data; applying a threshold thedifference frame to eliminate pixels therein below a specified intensityvalue; calculating a pixel size of a remaining blob in the differenceframe in a direction of blood flow; calculating a size of the remainingblob using the pixel size and a spatial resolution; and calculating avelocity by using a distance traveled by the remaining and a timebetween frames.

In at least one example, the method further includes: dividing themotion video data into frames each including pixels; identifying whichof the pixels receive some portion of the light-emitting marker using anintensity threshold; creating a logical frame in which a respectiveindicator for each pixel can be set as true or false; setting theindicators of the identified pixels as true; setting the indicators ofall other pixels as false; calculating a difference frame by subtractinga first logical frame from a second logical frame such that thedifference frame includes pixels that reached the specified thresholdafter a time of the first logical frame; finding length in pixels of theremaining blob in the difference frame in a direction of blood flow;converting the length in pixels of the difference frame to physicaldistance using the spatial resolution; and calculating velocity bydividing the physical distance by a time between frames.

According to at least one embodiment, a system for fluorescence basedtracking of a light-emitting marker in a bodily fluid stream includes: adelivery apparatus configured to provide a light-emitting marker into abodily fluid stream; a sensor configured to monitor a region of interesttraversed by the bodily fluid stream; and a computing device configuredto: record data generated by the sensor; determine time characteristicsof the recorded data; and calculate flow characteristics based on thetime characteristics.

In at least one example, the sensor includes a camera, and the recordeddata includes motion video data.

In at least one example, the computing device is further configured to:divide the motion video data into kernels; identify which of the kernelsreceive some portion of the light-emitting marker using an intensitythreshold; compute, for each identified kernel, an intensity signal dataset including information of mean light intensity versus time; performsmoothing on each intensity signal data set; calculate a lag timebetween the intensity signal data sets of neighboring identified kernelsusing cross-correlation; using a spatial resolution and the lag time,calculate velocity vectors; sum the velocity vectors of neighboringkernels to create a resultant velocity vector; and generate a velocitymap from the resultant velocity vectors for all kernels.

In at least one example, the computing device performs smoothing on eachintensity signal data set by time window averaging. In at least oneexample, the computing device performs smoothing on each intensitysignal data set by using a Gaussian filter.

In at least one example, the computing device is further configured to:divide the motion video data into kernels; identify which of the kernelsreceive some portion of the light-emitting marker using an intensitythreshold; compute, for each identified kernel, an intensity signal dataset including information of mean light intensity versus time; performsmoothing on each intensity signal data set; for each particularidentified kernel, find segments in which a slope of the intensitysignal data set rises for a minimum consecutive number of frames orfalls for a minimum consecutive number of frames, which segments occurwhen a leading edge or falling edge of a portion of the light-emittingmarker passes through the identified kernel; search the intensity signaldata sets of neighboring identified kernels for a rising or fallingsegment of similar length; calculate a lag time between segments in theparticular identified kernel and segments in the neighboring identifiedkernels; use a spatial resolution and the lag time to calculate velocityvectors; sum the velocity vectors of neighboring kernels to create aresultant velocity vector; and generate a velocity map from theresultant velocity vectors for all kernels.

In at least one example, the computing device is further configured to:calculate a difference frame by subtracting a frame of the motion videodata from a consecutive frame of the motion video data; apply athreshold the difference frame to eliminate pixels therein below aspecified intensity value; calculate a pixel size of a remaining blob inthe difference frame in a direction of blood flow; calculate a size ofthe remaining blob using the pixel size and a spatial resolution; andcalculate a velocity by using a distance traveled by the remaining bloband a time between frames.

In at least one example, wherein the computing device is furtherconfigured to: divide the motion video data into frames each includingpixels; identify which of the pixels receive some portion of thelight-emitting marker using an intensity threshold; create a logicalframe in which a respective indicator for each pixel can be set as trueor false; set the indicators of the identified pixels as true; set theindicators of all other pixels as false; calculate a difference frame bysubtracting a first logical frame from a second logical frame such thatthe difference frame includes pixels that reached the specifiedthreshold after a time of the first logical frame; find length in pixelsof the remaining blob in the difference frame in a direction of bloodflow; convert the length in pixels of the difference frame to physicaldistance using the spatial resolution; and calculate velocity bydividing the physical distance by a time between frames.

BRIEF DESCRIPTION OF THE DRAWINGS

The previous summary and the following detailed descriptions are to beread in view of the drawings, which illustrate particular exemplaryembodiments and features as briefly described below. The summary anddetailed descriptions, however, are not limited to only thoseembodiments and features explicitly illustrated.

FIG. 1 shows a fluorescent marker delivery time plot in a fluid streamand a corresponding response time plot of light intensity measureddownstream in a fixed field of view according to at least oneembodiment.

FIG. 2 shows a flowchart representing a method, according to at leastone embodiment, of generating a velocity vector map using a crosscorrelation technique.

FIG. 3 shows a flowchart representing a method, according to at leastone embodiment, of generating a velocity vector map using leading andfalling edge considerations.

FIG. 4 shows a flowchart representing a method, according to at leastone embodiment, of generating a velocity vector map using subtraction.

FIG. 5 shows a flowchart representing a method, according to at leastone embodiment, of generating a velocity vector map using thresholding.

FIG. 6 shows a system, according to at least one embodiment, by which atleast the methods of FIGS. 2-5 are implemented.

DETAILED DESCRIPTIONS

These descriptions are presented with sufficient details to provide anunderstanding of one or more particular embodiments of broader inventivesubject matters. These descriptions expound upon and exemplifyparticular features of those particular embodiments without limiting theinventive subject matters to the explicitly described embodiments andfeatures. Considerations in view of these descriptions will likely giverise to additional and similar embodiments and features withoutdeparting from the scope of the inventive subject matters. Although theterm “step” may be expressly used or implied relating to features ofprocesses or methods, no implication is made of any particular order orsequence among such expressed or implied steps unless an order orsequence is explicitly stated.

Any dimensions expressed or implied in the drawings and thesedescriptions are provided for exemplary purposes. Thus, not allembodiments within the scope of the drawings and these descriptions aremade according to such exemplary dimensions. The drawings are not madenecessarily to scale. Thus, not all embodiments within the scope of thedrawings and these descriptions are made according to the apparent scaleof the drawings with regard to relative dimensions in the drawings.However, for each drawing, at least one embodiment is made according tothe apparent relative scale of the drawing.

Fluorescence based tracking according to several embodiments describedherein includes the providing of a marker such as a glowing dye into afluid stream, such as a bloodstream, and making measurements andgenerating imagery based on the arrival, movement, and departure of themarker downstream as detected by sensor(s) to characterize the flow ofthe fluid stream and vessels or structures within which the flowtravels. The marker is provided into a fluid stream for example bydirect injection or via a port as discrete bolus deliveries separatedover time. A bolus refers to the administration of a discrete amount ofa fluid substance, in this case the marker into a bodily fluid streamsuch as blood, in order to provide a concentration of the substance togain a response. A bolus can be delivered by active pumping or bypassive gravity based delivery such as via an intravenous drip line. Inat least one embodiment, a central line delivery arrangement is used, inwhich a port is placed in fluid communication with the subclavian veinand bolus deliveries are injected into the port. The dye brieflyfluoresces when excited by an illumination source that emits aparticular range of wavelengths. The dye is illuminated over the Regionof Interest (ROI) where imaging of the fluorescence is also performed.

A field of view monitoring is established by placement of a sensor, suchas a high speed camera, at a region of interest. The field of view canbe established and held generally fixed as the marker enters andtraverses the field of view of a high-speed camera sensitive to thelight emitted by the marker. Time characteristics of the light output ofthe marker traversing the field of view can be deducted from the lightoutput intensity as recorded by the camera. A field of view may beestablished for example at the heart or other organ where flowdiagnostics are wanted.

The visual response in the field of view indicates presence of themarker, with the intensity of the light response being correlated withthe time evolving concentration of the marker in the stream as themarker diffuses and travels with the host fluid. The light intensity inthe field of view may typically have both rise and fall characteristics.The rise characteristics correspond to the arrival and increasingconcentration of the marker in the field of view. The fallcharacteristics correspond to the departure or diffusion of the markerand/or the reduction of its light output. In the case of a dye marker ina blood stream as injected by a bolus, rise time may be faster generallythan fall time such that response time curves typically show steeperclimbs than falls.

FIG. 1 shows a fluorescent marker delivery time plot 102 in a fluidstream and a corresponding response time plot 112 of light intensitymeasured downstream in a fixed field of view according to at least oneembodiment. The plots 102 and 112 are shown on a common time axis 100.The delivery time plot 102 records several bolus deliveries 104 as stepfunctions separated in time. The response plot 112 records thecorresponding response functions 114 of light intensity in a field ofview downstream from the marker delivery location into a fluid stream.Each response function 114 is shown as a wave or envelope having a riseside 116 representing the arrival of the marker in the field of view,and a fall side 118 indicating the departure or diffusion of the markerand/or the reduction of its light output. In order to correlate bolusdeliveries and data acquisition, a marker delivery system in at leastone embodiment includes a controller and a delivery pump in wired orwireless communication. A data acquisition system including the cameraand a computing device for recording, analyzing and visualizing the dataand/or field of view are also in communication with the controller in atleast one embodiment.

The time of initiation, delivered volume, and duration of each bolusdelivery can be controlled. The time interval between consecutive bolusdeliveries is also controlled. Thus, multiple parameters for bolusdelivery can be adjusted to ultimately query and determine varied flowcharacteristics within a region of interest subjected to field of viewmonitoring. Shorter time gaps can be used for slower moving fluids andlonger time gaps can be used for faster moving fluids within the regionof interest. Injection times can be varied to address differentanatomical depths and tissue surface barriers.

In at least one embodiment, as the marker from a bolus delivery entersan established field of view, the light response of a bolus is capturedby a high speed camera. The time domain dynamics of the light responseis analyzed to arrive at velocity vectors representing movement of thehost fluid in the field of view. Several embodiments of generatingvelocity vectors using the data from fluorescence based tracking aredescribed in the following with reference to FIGS. 2-5. In each, a videoincluding multiple frames (images) taken of a region of interest isanalyzed to track pixels in or across the field of view.

A method 200 of generating a velocity vector map using a crosscorrelation technique, according to at least one embodiment, isrepresented as a flow chart in FIG. 2. In step 202, divide the videointo spatiotemporal cubes, which are termed “kernels” in thesedescriptions. In step 204, which is optional, isolate kernels in whichfluorescence appears at any point during the length of the video usingan intensity threshold. In step 206, compute a signal for each kernel ofmean intensity vs. time. The signal may be 1D, 2D, or 3D. Vessel(s) maycross under other vessel(s) and at different angles, and multipleperspectives from different cameras can be used. The potential use ofcoded apertures is within the scope of these descriptions. In step 208,which is optional, perform smoothing on the intensity signal for eachkernel using time window averaging, Gaussian filter, etc. In step 210,for each kernel, calculate the lag time between the intensity signal ofthe kernel and the time intensity signals of its neighboring kernelsusing cross-correlation. In step 212, using the known spatial resolutionof the image, convert lag time in each direction into velocity. In step214, sum the resulting velocity vectors to each neighboring kernel tocreate one resultant velocity vector. In step 216, generate a velocitymap from the resultant velocity vectors for all kernels.

A method 300 of generating a velocity vector map using leading andfalling edge considerations, according to at least one embodiment, isrepresented as a flow chart in FIG. 3. In step 302, divide video intokernels. In step 304, which is optional, isolate kernels in whichfluorescence appears at any point during the length of the video usingan intensity threshold. In step 306, compute a signal for each kernel ofmean intensity vs. time. As described above with reference to step 206of FIG. 2, the signal may be 1D, 2D, or 3D. In step 308, which isoptional, perform smoothing on the intensity signal for each kernelusing time window averaging, Gaussian filter, etc. In step 310, for eachkernel, find segments in which the slope of the intensity signal risesfor a minimum consecutive number of frames or falls for a minimumconsecutive number of frames. These segments occur when the leading orfalling edges of the ICG (fluorescein or other glow dye) bolus passthrough the kernel. In step 312, search the intensity signals ofneighboring kernels for a rising or falling segment of similar length.In step 314, calculate the temporal offset (lag time) between thesegment in the original kernel and the segment in the neighboringkernels. In step 316, using the known spatial resolution of the image,convert lag time in each direction into velocity. In step 318, sum theresulting velocity vectors to each neighboring kernel to create oneresultant velocity vector. In step 320, generate a velocity map from theresultant velocity vectors for all kernels.

A method 400 of generating a velocity vector map using subtraction,according to at least one embodiment, is represented as a flow chart inFIG. 4. In step 402, subtract two consecutive frames, resulting in adifference frame. In step 404, threshold difference frame to eliminatepixels below a specified intensity value. In step 406, calculate pixelsize of a remaining blob in the difference frame in the direction ofblood flow. In step 408, convert size of blob in pixels to physicaldistance using spatial resolution. In step 410, calculate velocity bydividing distance traveled by time between frames.

A method 500 of generating a velocity vector map using thresholding,according to at least one embodiment, is represented as a flow chart inFIG. 5. In step 502, for each frame in video, isolate pixels withintensities above specified threshold. Create logical frame and set allpixels at or above threshold to true. Set all other pixels to false. Instep 504, for two consecutive frames, subtract the first logical framefrom the second logical frame. The resulting frame contains the pixelsthat reached the specified threshold between frames. In step 506, findthe length in pixels of the remaining blob in the difference frame inthe direction of blood flow. In step 508, convert the pixel length ofthe difference frame to physical distance using the spatial resolution.In step 510, calculate velocity by dividing physical distance by thetime between frames.

FIG. 6 shows a system 600, according to at least one embodiment, bywhich at least the methods of FIGS. 2-5 are implemented. The system 600includes a computing device 602, a delivery apparatus 604 configured toprovide a light-emitting marker 606 into a bodily fluid stream, and asensor 610 configured to monitor a region of interest traversed by thebodily fluid stream. The computing device 602 records data generated bythe sensor 610, determines time characteristics of the recorded data;and calculates flow characteristics based on the time characteristics.

The computing device 602 is illustrated as a laptop or other personalcomputer. Other computing devices including local and remote servers arewithin the scope of these descriptions. The delivery apparatus 604provides a light-emitting marker 606. The delivery apparatus 604 is incommunication with and/or under the control of the computing device 602.The delivery apparatus 604 may include a powered pump or a gravity basedarrangement. The light-emitting marker 606 may be delivered to thebodily fluid stream 620 via a catheter, an intravenous drip line, acentral line delivery, or other needle or port device. The deliveryapparatus 604 delivers the light-emitting marker 606 in discrete bolusdeliveries separated over time. The light-emitting marker 606 mayinclude Indocyanine green (ICG), Fluorescein or other glow dye. Two ormore dyes, each having a different respective color, may be used. Forexample, each bolus of two or more may include a different dye and thusthe presence and response of each can be determined separately by colordistinction.

The bodily fluid stream 620 in FIG. 6 is represented as flowing along ablood vessel or other biological conduit 622. Several downstream organsor tissue areas 624, 626 and 628 are represented. By placement andselection of field of view of the sensor 610, a region of interest canbe selected for observation downstream of the bodily fluid stream 620carrying with it the light-emitting marker 606. The sensor 610 monitorsfor fluorescence or other indication of the presence of thelight-emitting marker 606 in the selected field of view. The sensor 610can be a high-speed and high-resolution camera for example.

Several fields of view are represented. In a first exemplary field ofview 630, the sensor 610 observes an area where the bodily fluid stream620 is divided into several downstream flows. In a second exemplaryfield of view 632, the sensor 610 observes an area downstream of thedivision to isolate monitoring to a single branch of downstream flow. Ina third exemplary field of view 634, the sensor 610 observes aparticular organ or tissue area 628. These examples represent that auser such as a physician can deliver a light-emitting marker 606 at anyselected location and then observe the time evolving arrival anddispersion or other activity of the marker downstream of the selectedlocation at any selected field of view. In at least one embodiment, acentral line delivery arrangement is used, in which a port is placed influid communication with the subclavian vein and bolus deliveries of thelight-emitting marker 606 are injected into the port.

The delivery apparatus 604 and sensor 610 are shown as connected to thecomputing device 602 by respective cables 612 and 614, however wirelessconnections may be used as well. The light-emitting marker 606 brieflyfluoresces when excited by an illumination source 640 that emits aparticular range of wavelengths upon the region of interest within thefield of view of the sensor 610. The illumination source 640 is alsoshown as connected to the computing device 602 by a cable 642, however awireless connection may be used as well. The computing device correlatesactivations of the delivery apparatus 604, the illumination source 640,and the sensor 610, and collects data from the sensor 610 as thelight-emitting marker 606 in the field of view responds to theexcitation from the illumination source 640.

In various embodiments, the computing device 602 is configured to recorddata generated by the sensor 610; determine time characteristics of therecorded data; and calculate flow characteristics based on the timecharacteristics. Further embodiments and examples of fluorescence basedimaging and data analysis conducted by the computing device 602 aredescribed above with reference to FIGS. 2-5.

Particular embodiments and features have been described with referenceto the drawings. It is to be understood that these descriptions are notlimited to any single embodiment or any particular set of features, andthat similar embodiments and features may arise or modifications andadditions may be made without departing from the scope of thesedescriptions and the spirit of the appended claims.

What is claimed is:
 1. A system for fluorescence based tracking of alight-emitting marker in a bodily fluid stream, the system comprising: adelivery apparatus configured to provide a light-emitting marker intothe bodily fluid stream; a camera configured to monitor a region ofinterest traversed by the bodily fluid stream; and a computing deviceconfigured to: record motion video data generated by the camera;determine time characteristics of the recorded data; and calculate flowcharacteristics based on the time characteristics.
 2. The systemaccording to claim 1, wherein the computing device is further configuredto: divide the motion video data into kernels; identify which of thekernels receive some portion of the light-emitting marker using anintensity threshold; compute, for each identified kernel, an intensitysignal data set comprising information of mean light intensity versustime; perform smoothing on each intensity signal data set; and calculatea lag time between the intensity signal data sets of neighboringidentified kernels using cross-correlation.
 3. The system according toclaim 1, wherein the computing device is further configured to: using aspatial resolution and the lag time, calculate velocity vectors; sum thevelocity vectors of neighboring kernels to create a resultant velocityvector; and generate a velocity map from the resultant velocity vectorsfor all kernels.
 4. The system according to claim 3, wherein thecomputing device performs smoothing on each intensity signal data set bytime window averaging or by using a filter.
 5. The system according toclaim 3, wherein the computing device is further configured to: for eachparticular identified kernel, find segments in which a slope of theintensity signal data set rises for a minimum consecutive number offrames or falls for a minimum consecutive number of frames, whichsegments occur when a leading edge or falling edge of a portion of thelight-emitting marker passes through the identified kernel; search theintensity signal data sets of neighboring identified kernels for arising or falling segment of similar length; and calculate a lag timebetween segments in the particular identified kernel and segments in theneighboring identified kernels.
 6. The system according to claim 3,wherein the computing device is further configured to: calculate adifference frame by subtracting a frame of the motion video data from aconsecutive frame of the motion video data; apply a threshold to thedifference frame to eliminate pixels therein below a specified intensityvalue; calculate a pixel size of a remaining blob in the differenceframe in a direction of bodily fluid flow; calculate a size of theremaining blob using the pixel size and a spatial resolution; andcalculate a velocity by using a distance traveled by the remaining bloband a time between frames.
 7. The system according to claim 3, whereinthe computing device is further configured to: create a logical frame inwhich a respective indicator for each pixel can be set as true or false;set the indicators of the identified pixels as true; set the indicatorsof all other pixels as false; calculate a difference frame bysubtracting a first logical frame from a second logical frame such thatthe difference frame comprises pixels that reached the specifiedthreshold after a time of the first logical frame; find length in pixelsof the remaining blob in the difference frame in a direction of bodilyfluid flow; convert the length in pixels of the difference frame tophysical distance using the spatial resolution; and calculate velocityby dividing the physical distance by a time between frames.
 8. A methodof fluorescence based tracking of a light-emitting marker in a fluidstream, the method comprising: monitoring, with a camera, a region ofinterest traversed by fluid stream into which a light-emitting markerhas been introduced; recording motion video data generated by thesensor; dividing the motion video data into frames each comprisingpixels; identifying which of the pixels receive some portion of thelight-emitting marker using an intensity threshold; calculating adifference frame by subtracting a frame of the motion video data from aconsecutive frame of the motion video data; and applying a threshold tothe difference frame to eliminate pixels therein below a specifiedintensity value.
 9. The method according to claim 8, further comprising:calculating a pixel size of a remaining blob in the difference frame ina direction of fluid flow; calculating a size of the remaining blobusing the pixel size and a spatial resolution; and calculating avelocity by using a distance traveled by the remaining blob and a timebetween frames.
 10. The method according to claim 9, further comprising:creating a logical frame in which a respective indicator for each pixelcan be set as true or false; setting the indicators of the identifiedpixels as true; setting the indicators of all other pixels as false;calculating a difference frame by subtracting a first logical frame froma second logical frame such that the difference frame comprises pixelsthat reached the specified threshold after a time of the first logicalframe; finding length in pixels of a remaining blob in the differenceframe in a direction of fluid flow; converting the length in pixels ofthe difference frame to physical distance using the spatial resolution;and calculating velocity by dividing the physical distance by a timebetween frames.